Background: We have shown that extract from Agaricus bisporus, the common white button mushroom (WBM), contains phytochemicals that suppress aromatase activity, inhibit breast cancer (BC) cell proliferation, and decrease mammary tumor formation in vivo. We now report a translational clinical trial to determine the optimal dose to induce this effect in humans. Methods: WBM were freeze-dried and pressed into a tablet containing 500mg of powdered extract, each roughly equivalent to 5g of whole WBM. Postmenopausal women diagnosed with BC at least 5 years prior, who were at least 3 months off therapy and recurrence-free, were treated with a 12-week course of 5, 8, 10, or 13g of mushroom extract daily. Aromatase inhibition was evaluated with serial serum sex steroid hormone and ex vivo plasma aromatase activity (AA) assays. Response was defined as a ≥50% decrease in free estradiol (FE2). Because immunologic effects have been reported with medicinal mushrooms, cytokines were also measured. Results: WBM extract up to 13g per day was well tolerated, with a median adherence of 95%, 96%, 95%, and 98% on the four escalating doses, respectively (n=6 each). Although no patients met the pre-defined response criterion, FE2 was observed to trend upward over the 12-week treatment course for the 5g and 8g dose groups and remain stable among the 10g and 13g dose groups (p=.05). AA measurements were able to detect a consistent post-prandial peak in activity in the 5g and 8g dose groups (p=.003 and <.001, respectively), which diminished in the 10g group and disappeared in the 13g group. Among patients with detectable baseline cytokine levels, IL-1β, IL-2, and IL-6 decreased on treatment, but there was no clear dose effect. Conclusions: High doses of WBM extract are well tolerated. An ex vivo assay of aromatase activity developed in our lab is sensitive to short-term changes and demonstrated suppression of post-prandial fluctuations with a 10g or 13g daily dose of WBM extract. These results suggest that anti-aromatase phytochemicals are present in plasma with daily consumption of 100-130g whole WBM, but not at high enough concentrations to significantly reduce estrogen levels from baseline in 12 weeks.